Although disorders of iron metabolism such as iron deficiency and iron overload are common in humans, our understanding of the proteins that mediate iron uptake into cells remains incomplete. In the first cycle of this grant, we established that the transmembrane protein ZIP14, a member of the ZIP family of metal-ion transporters, could transport iron into cells and is up-regulated by iron overload in the liver and pancreas, tissues that are particularly susceptible to iron-related pathologies. A direct role for ZIP14 in ion metabolism in vivo has been revealed by our recent studies of ZIP14 knockout mice, which display alterations in iron status parameters and a marked impairment in the uptake of non-transferrin-bound iron by the liver and pancreas. By screening other ZIP family members for iron transport activity, we recently discovered that ZIP8, the most closely related protein to ZIP14, could also transport iron and is up-regulated by cellular iron loading. In the next funding cycle, we will define the physiologic roles of ZIP14 and ZIP8 in iron metabolism. Our central hypothesis is that ZIP14 and ZIP8 participate in iron uptake by various tissues, including those that accumulate iron during iron overload. The first aim of the proposed research will define the role of ZIP14 in tissue iron loading by using ZIP14 knockout mice intercrossed with Hfe and Hjv knockout mice, mouse models of the human iron overload disorders, hereditary hemochromatosis and juvenile hemochromatosis, respectively. In our second aim, we will determine the mechanism of impaired hepatic iron loading in double-knockout ZIP14;Hfe mice by examining hepatic uptake of non-transferrin-bound iron and transferrin-bound iron. In our third aim, we will elucidate the role(s) of ZIP8 in iron metabolism and the contribution of ZIP8 to iron loading of the pancreas and heart by using novel tissue-specific ZIP8 knockout mice. The work proposed in these aims will help to close the gap in our mechanistic understanding of how tissues take up iron. Such results are expected to have a positive impact because they may identify new therapeutic targets for treating disorders of iron metabolism and their associated pathologies.